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Article
Laura Filkins, Jeffrey A SoRelle, John Schoggins, and Jason Y Park. Laboratory Action Plan for Emerging SARS-CoV-2 Variants Clin Chem 2021; 67:5 720–23.
Guest
Dr. Laura Filkins is an assistant professor of pathology at the University of Texas Southwestern Medical Center and director of microbiology at Children’s Health in Dallas.
Transcript
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Bob Barrett:
This is a podcast from Clinical Chemistry, sponsored by the Department of Laboratory Medicine at Boston Children’s Hospital.; I’m Bob Barrett.
In late 2020, a clinically significant SARS-CoV-2 variant with an increased transmission rate was identified in the United Kingdom.; It has subsequently been identified in multiple countries throughout the world and continues to spread unabatedly.; So far, such variants appear to be detectable by commonly used nucleic acid-based tests and hopefully will not present a diagnostic challenge for clinical laboratories.; As the COVID-19 pandemic continues, we anticipate additional clinically significant SARS-CoV-2 variants will emerge.
An opinion piece in the May 2021 issue of Clinical Chemistry proposes several preparatory actions for clinical laboratories to deal with emerging SARS-CoV-2 variants.; The lead author of that paper is Dr. Laura Filkins, an assistant professor of pathology at the University of Texas Southwestern Medical Center and director of microbiology at Children’s Health in Dallas, and Dr. Filkins is our guest in this podcast.
Doctor, it seems that every day on the news we learn of a new or different variant of SARS-CoV-2.; Let’s get basic first off.; What exactly is a variant of virus and how common are they?
Laura Filkins:
A variant of a virus is a virus whose genome sequence is different than that of a reference virus.; So, in the case of SARS-CoV-2, which is the virus that causes coronavirus disease 2019 or COVID-19, we usually use one of the earliest sequenced viruses from a patient in Wuhan at the end of 2019 as being the reference.; Therefore, any change in the virus or anything different from that original reference is considered a variant, and at this point in the pandemic, essentially every virus of SARS-CoV-2 is a variant, so it’s very common, and I should also say that variants are not unique to SARS-CoV-2.; Every virus in every organism has variants.
Bob Barrett:
So, how are such variants detected and identified?
Laura Filkins:
Good question.; So, variants are identified a little bit differently than most of our standard laboratory testing for SARS-CoV-2.; So, to back up a step, most of our laboratory-based tests that we perform to diagnose SARS-CoV-2 infection are amplification-based assays, like PCR, and we can determine, yes, a sample contains SARS-CoV-2 or, no, SARS-CoV-2 is not detectable, but to identify a new variant or even a known variant, PCR isn’t quite enough.
So, we use whole genome sequencing for this.; We typically use a next-generation sequencing platform and then pair that with bioinformatics tools, and really amazingly, with some of these newer technologies, we can actually identify variants within potentially a few hours after collecting a sample.; Now, realistically with the workflows and most laboratories, that timeline is usually closer to a couple of days or maybe even a week but still, that’s within the turnaround time that could be clinically actionable.
Bob Barrett:
Do all viral variants impact clinical conditions and/or public health?
Laura Filkins:
Actually, no. many viral various appear to have no effect on clinical or public health.; Some variants actually make the virus less fit and those typically don’t stay around for very long, but some variants of course do have a significant clinical impact and the variants that we are most alert for in the lab and in the clinical setting would be those variants that appear to affect viral fitness, either by changing its replication or infectivity, transmission, potentially even host interactions that could lead to more severe disease, and we also look for viruses that are more resistant to the therapies that we have available or potentially allow the virus to evade a patient’s immune response after vaccination.
So, to address these different degrees of impact, we use the classification system which includes variants of interest, meaning those we’re studying, and variants of concern meaning those with mutations that appear to confer benefits to the virus that are likely to impact clinical and public health, and currently there are four variants of concern that are recognized by the World Health Organization.; Those go by both a few different scientific lineage-based names and also by, more recently, their WHO label, which is Alpha, Beta, Gamma, and Delta, you’ve probably heard of in the news recently, and then the U.S. CDC actually includes two additional variants in concern, both of which are epsilon.
Bob Barrett:
So, could the presence of viral variants in our population impact the ability for labs to detect COVID-19 infection?
Laura Filkins:
Yes.; Variation of a virus can impact the ability for us to detect SARS-CoV-2 using our current clinical diagnostic tests, and this is actually one of my greatest fears as a laboratory director.
The primary concern is that variation could lead to false negative results if the wrong combination of mutations occur, and of course the risk with false negatives is that we may then have a patient who altered their behavior because of that negative result, and this could mean acting differently in public or even in the private setting.; Another potential impact of that is using less PPE or protective equipment in the healthcare setting, and this would be done based on trusting that the patient really does not have SARS-CoV-2 infection.
So, in order to avoid these potential false results, there are a few things that in the laboratory we can do.; The first is to consistently monitor for reports of local variants and even potentially global variants that haven’t entered your local population yet, and we want to make sure that our COVID tests in the lab can detect those variants.; So, how we can go about this, first, always review the manufacturer’s report to see if there are any predicted performance issues that the manufacturer is reporting.; Of course, it’s a little bit challenging because many of the designs of commercial assays are proprietary and we really do rely on the manufacturers for that, but for tests whose design is public, labs can perform their own in silico predictions to determine if the reagents are anticipated to find them and detect the virus variant.
Ideally, labs would go one step further whenever possible and if there are available reagents to do so, and beyond just in silico predictions we would like to test in vitro, whether or not our assays can detect variants of virus.; So, we can use either whole virus or recombinant virus that mimics variants and determine if our platforms qualitatively can detect the virus, and if there’s any impact on limit of detection.
Bob Barrett:
Is there a need for broad clinical application for variant identification?
Laura Filkins:
This is a challenging question to answer because the word “need” carries some degree of opinion with it, but currently I think it’s fair to say that sequencing is imperative to public health.; However, clinical care is independent of variant identification in most cases at the moment, and really knowing if the patient is infected with SARS-CoV-2 or not but not necessarily knowing which variant, will drive the majority of clinical decision-making.; On the public health side though, the amount of virus sequencing that has been performed even in the first half of 2021 is exponentially increased compared to that earlier in the pandemic, and monitoring changing epidemiology will be crucial over the coming months.
However, sequencing is resource-intensive and I think it’s unrealistic at this point to expect routine variant identification for all positive samples.; On the other hand, screening for sequence determinants may become more routine as we progress through the pandemic and there are few potential clinical applications for this such as detecting known variants that could have reduced susceptibility to monoclonal antibody therapies, differentiating between persistent infection in a patient from a single virus versus reinfection, or potentially detecting genetic changes that confer resistance or reduce susceptibility after vaccination.
Bob Barrett:
So, how can an individual laboratory test for known variants?
Laura Filkins:
As we’ve discussed, whole genome sequencing is the primary method and to confirm that a virus belongs to a given lineage, let’s say B.1.1.7 or the alpha variant, the entire genome must be determined.; However, for labs that don’t plan to perform whole genome sequencing, there are several screening methods that I alluded to, and these methods usually look for a change or multiple changes that are consistent with a given lineage, but not the definitive for that lineage.
So, some screening methods include partial genome or partial gene sequencing.; We also have several amplification-based methods, and these are typically assays that are designed so that the primers of the probe positively bind to a change in the sequence that’s consistent with that variant.; Although, we can also use the reverse which is to have a drop out of a signal when a variant occurs, we can do melt curve analysis to predict sequence changes within an amplicon, and there are also a few commercially designed and manufactured assays that have been built to detect known sequence variation, most of them geared towards screenings for the common variants of concern currently circulating.; I should note, though, that the commercial assays that currently are available do not have emergency use authorization from the FDA for variant detection.
Bob Barrett:
Well, it appears then there are increasingly available approaches to detect both novel and known variants for laboratories that don’t plan on performing whole genome sequencing or implementing screening assays specific to known nucleotide polymorphisms.
Are there other approaches to monitor potential variants?
Laura Filkins:
Yes.; There are several things that labs can do to monitor for variants without implementing additional testing.; My first recommendation is to monitor your institutional positivity rate and continuously compare those rates to local, regional, or even state-level epidemiology.; The goal of this comparative monitoring would be to look for an anomaly.; So, for example, if your institution’s positivity rates typically coincide with that of the county but then suddenly drop or spike relative to your comparator, your county, then that would be an opportunity for investigation.
Another approach applies specifically to labs that use molecular assays with certain features.; So, these are specifically tests that have multiple gene targets and provide a numeric signal for each target.; This is usually a cycle threshold, but depends on the platform. And using these assays, labs can perform target detection comparison plotting to look for atypical differences between the detection of the multiple targets.; That’s a little bit of a mouthful so, for example: let’s say you have an assay with an N gene and an S gene target. And most samples, most positive samples, will give a CT value for the N gene and the S gene that are about within three cycles of one another, and then you go about testing all of your patient samples, and you see that one of the samples, the N gene dropped out, or there’s an atypical difference between the level of detection between the N and the S gene.; Let’s say 10 CTs instead of your typical 3 CTs.
So, this would again be an opportunity to say, “I think there’s something unique about this sample,” and then investigate it further. Beyond lab results, there are some clinical scenarios that could be indicators for further assessment.; This usually requires collaboration between infection, prevention, and control, maybe your occupational health teams or your clinical teams, and in these situations what we look for are patients that merit further investigation when they have a positive sample.; This could be positive samples from a patient who is fully vaccinated or a patient who has been positive for a very prolonged period of time.
And then, finally labs can consider reaching out to their public health lab to inquire about what the public health needs are.; For example, you know, maybe your public health lab is looking for a subset of samples from multiple different hospitals, and I would just encourage that you ask your public health lab what is it that you can do to help facilitate their surveillance effort.
Bob Barrett:
That was Dr. Laura Filkins, an assistant professor of pathology at the University of Texas Southwestern Medical Center and director of microbiology at Children’s Health in Dallas.; She has been our guest in this podcast on “Laboratory Action Plans for Emerging SARS-CoV-2 Variants.”; That article appears in the May 2021 issue of Clinical Chemistry.; I’m Bob Barrett. Thanks for listening.